PCNA Complex and Implications for Clamp Sliding During DNA Replication and Repair

ARTICLE Received 6 Jun 2014 | Accepted 29 Jan 2015 | Published 12 Mar 2015 DOI: 10.1038/ncomms7439 Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair Alfredo De Biasio1,w, Alain Ibań˜ez de Opakua1, Gulnahar B. Mortuza2,3, Rafael Molina2, Tiago N. Cordeiro4, Francisco Castillo5, Maider Villate1, Nekane Merino1, Sandra Delgado1, David Gil-Cartoń1, Irene Luque5, Tammo Diercks1, Pau Bernado´4, Guillermo Montoya2,5 & Francisco J. Blanco1,6 The intrinsically disordered protein p15PAF regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15PAF–PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15PAF tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15PAF also contacts the inside of, and passes through, the PCNA ring. The disordered p15PAF termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15PAF binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15PAF acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding. 1 Structural Biology Unit, CIC bioGUNE, Parque Tecnolo´gico de Bizkaia Edificio 800, 48160 Derio, Spain. 2 Structural Biology and Biocomputing Programme, Centro Nacional de Investigaciones Oncolo´gicas, Melchor Fernańdez Almagro 3, 28029 Madrid, Spain. 3 Protein Structure and Function Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. 4 Centre de Biochimie Structurale, INSERM U1054, CNRS UMR 5048, Universite´ Montpellier 1 and 2, 29 rue de Navacelles, 34090 Montpellier, France. 5 Department of Physical Chemistry and Institute of Biotechnology, Universidad de Granada, Fuentenueva s/n, 18071 Granada, Spain. 6 IKERBASQUE, Basque Foundation for Science, MarıáDıáz de Haro 3, 48013 Bilbao, Spain. w Present address: Elettra Sincrotrone Trieste, Trieste, Italy. Correspondence and requests for materials should be addressed to A.De.B (email: [email protected]) or to F.J.B. (email: [email protected]). NATURE COMMUNICATIONS | 6:6439 | DOI: 10.1038/ncomms7439 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7439 he DNA-sliding clamp proliferating cell nuclear antigen binding to equivalent sites in the PCNA trimer, with a dissocia- (PCNA) is an essential factor in replication and repair that tion constant of 1.1 mMat25°C (Fig. 1c). Thus, three p15 Trecruits polymerases and other DNA-modifying enzymes molecules bind to the three PCNA protomers, with no indication to the replication fork1. PCNA forms an 86-kDa homotrimeric of cooperativity. The enthalpic contribution to the free energy ring2 that encircles the DNA and anchors binding partners, is large, but so is the entropic cost, in line with a loss in p15 preventing them from falling off the genomic template. The ring conformational freedom on binding to PCNA. has an inner diameter of B35 Å, much wider than the B24 Å As a disordered protein, p15 yields NMR spectra with sharp diameter of a DNA duplex in the B-form3. Apart from DNA- signals and poor dispersion in the proton frequency. On addition editing factors, PCNA also interacts with non-enzymatic proteins of excess PCNA, signal intensities decrease generally and most that regulate cell cycle control and survival4,5. Many PCNA- notably for the central residues V53-D75 that even disappear interacting proteins bind through the PIP-box, a consensus (Fig. 1d). This observation highlights the central region of p15 as sequence with the pattern QXXhXXaa, where h is an aliphatic- the principal binding site for PCNA, which includes the PIP-box hydrophobic residue, a aromatic-hydrophobic and X any motif (62QKGIGEFF69, conserved residues underlined). The p15 residue6. In all known co-crystal structures, the PIP-box NMR signals still visible in the complex retain their low residues bind to the PCNA front-face forming a short amino- dispersion, indicating that they remain largely disordered and terminal (N-terminal) b-strand that interacts with the PCNA flexible. Nevertheless, in the presence of PCNA, two segments in C-terminus, a short 310 helix placed in a hydrophobic pocket and the N- and C- terminal regions of p15 around the conserved basic a carboxyl-terminal (C-terminal) b-strand of variable length that residues R14K15 and P100, respectively, show significantly 7 15 pairs with the PCNA interdomain connecting loop (IDCL) . reduced signal intensities (Fig. 1d) and increased NR2 The recognition surface on PCNA is small, contacting 11–18 relaxation rates (Fig. 1e). While all R2 rates increase due to the residues of the ligand. The regions flanking these tightly bound larger molecular size, their abnormal increase in these two regions residues are usually not visible in electron density maps, indicates some conformational restrictions in the complex. Both suggesting that the chain breaks away from the clamp surface. regions contain clusters of positively charged amino acids that A common feature of the PIP-box region in these proteins is its might interact transiently and unspecifically with the negatively disordered nature, providing an adaptable, yet specific contact site charged PCNA surface. In line with this, chemical shift whose affinity for PCNA can be tuned by subtle changes in the perturbations (CSP) here are the largest observed outside the sequence8. core binding site (Fig. 1f), but still so small that only weak The PCNA-associated factor p15 (p15PAF, hereafter named transient interaction is expected. Changes in the NMR spectrum p15) is a nuclear protein with a PIP-box9 and is involved in the of PCNA on p15 binding are extensive (Fig. 1b), more than seen regulation of DNA replication and cell cycle progression by when bound to a PIP-box fragment of p21CIP1 (hereafter named interacting with PCNA10–12. Overexpression of p15 correlates p21)18, suggesting a larger binding area for p15. with tumour progression and poor prognosis in a number of To study the role of different p15 regions in the interaction human cancers13–16. Evidence for p15–PCNA interaction comes with PCNA, we designed three p15 fragments: the N-terminal from yeast two-hybrid and co-immunoprecipitation experiments, deletion mutant p1532–111 (hereafter named p15DN) lacking but no structural detail of the direct interaction is known. We many of the residues experiencing reduced NMR signal intensity 15 50–77 have previously shown that p15 is a monomeric intrinsically and enhanced NR2 rates on PCNA binding; the p15 disordered protein with conformational preferences in certain fragment comprising the central residues that experience the regions, including the PIP-box17. largest drop in NMR signal intensity on PCNA binding; and Here we present an extensive characterization of the interac- p1559–70 that corresponds to the smallest PIP-box containing tion between p15 and PCNA based on an integrative structural fragment of p21 with reported binding to PCNA19. approach. Three p15 molecules bind to the trimeric PCNA ring All three p15 fragments bind PCNA, but with decreasing with low micromolar affinity. The central region of p15, including affinity (Supplementary Fig. 1 and Supplementary Table 1). The the PIP-box, is tightly bound to the front-face and inner surface N-terminal deletion mutant binds PCNA with only slightly lower of PCNA, while the N- and C-termini remain predominantly affinity than p15, implying that any transient interactions of disordered at its back- and front-face, respectively, with transient the N-terminal V2-S31 residues stabilize the complex only interactions at the PCNA outer surface. This arrangement is marginally. The affinity of p1550–77, however, is reduced by a consistent with the protection from degradation by the 20S factor of 5 and the shortest core fragment p1559–70 even by a proteasome particle, which rapidly degrades free p15. Most factor of 33. These results indicate that residues G50-T58 and/or importantly, we show that p15 binds DNA, mainly through its L71-E77 strongly contribute to PCNA binding, which is further histone-like N-terminal tail. While PCNA alone does not interact enhanced by residues A32-G49. with DNA, p15 binds to PCNA and DNA simultaneously via The NMR spectra of PCNA in the presence of the different independent sites. Molecular modelling and electron microscopy constructs indicate rather similar binding modes for p15, p15DN show that a ternary complex with both p15 and DNA inside the and p1550–77, but less so for p1559–70 (Supplementary Fig. 2). The PCNA ring can form. We discuss the possible implications of strong intensity reduction in many PCNA signals in the presence these findings for the regulation of DNA replication and repair, of p15 (and to a lesser extent in the presence of p15DN) most and propose that p15 acts as a flexible drag that regulates PCNA likely derives from line broadening caused by the increased size of sliding along the DNA, facilitating the switch from replicative to the complex (125 kDa) and by transient interactions with the p15 translesion synthesis polymerase binding. chain termini. While we have previously assigned the NMR spectrum of PCNA free and bound to a p21 PIP-box peptide18,20, p15 excess leads to prohibitively large intensity reduction and line Results broadening that precluded assignment of the p15-bound PCNA p15–PCNA interaction involves an extended PIP-box region. spectrum by triple resonance methods. Instead, we transferred the Changes in the NMR spectra of p15 and PCNA in the presence of assignment from free PCNA, based on extrapolation from signal each other (Fig.

Load more